Advances in Production of Hydroxycinnamoyl-Quinic Acids: From Natural Sources to Biotechnology

Hydroxycinnamoyl-quinic acids (HCQAs) are polyphenol esters formed of hydroxycinnamic acids and (-)-quinic acid. They are naturally synthesized by plants and some micro-organisms. The ester of caffeic acid and quinic acid, the chlorogenic acid, is an intermediate of lignin biosynthesis. HCQAs are biologically active dietary compounds exhibiting several important therapeutic properties, including antioxidant, antimicrobial, anti-inflammatory, neuroprotective, and other activities. They can also be used in the synthesis of nanoparticles or drugs. However, extraction of these compounds from biomass is a complex process and their synthesis requires costly precursors, limiting the industrial production and availability of a wider variety of HCQAs. The recently emerged production through the bioconversion is still in an early stage of development. In this paper, we discuss existing and potential future strategies for production of HCQAs.


Introduction
HCQAs (Table 1) are mostly produced in plants by ester formation of a hydroxycinnamic acid (primarily p-coumaric, caffeic, ferulic and sinapic acids) with a (-)-quinic acid from the phenylpropanoid pathway, and they are linked with lignin synthesis [1]. They belong to a large and diverse group of phenolic compounds, often termed as chlorogenic acids [2]. The extended list of chlorogenic acids, which contains approximately 400 compounds [2], also encompass several derivatives and isomers of quinic acid, including shikimic acid, its epimers, 4-deoxy-, muco-, methyl-and butyl-quinic acids esterified with hydroxycinnamic and hydroxybenzoic acids or some of their derivatives. The nomenclature and trivial names of compounds of chlorogenic acids are rather complicated and they are explained in [3].
Here, we primarily focus on the HCQAs (Table 1). Amongst these, the most abundant and important compound is 5-caffeoylquinic acid (5-CQA), often referred to as chlorogenic acid. In 2018, the market size of 5-CQA was 130 million and it is expected to reach 150 million US$ by 2025 [4].
Over the last decade, a wide interest in HCQAs has been reflected by an exponentially growing number of scientific publications ( Figure 1). However, the main focus has been on HCQAs extraction from plants and to some extent on their chemical synthesis, whereas studies on microbial production of these acids have been limited.
Moreover, 5-CQA has found applications in the synthesis of metals' nanoparticles as a reducing and stabilizing agent [28,29]; in the production modification of carbon ceramic electrode for NADH detection [30]; in red food dye preparation from coupling of tryptophan and 5-CQA [31]; and the preservation of food by preparing the edible coating with chitosan [32]. This compound is also referred to as the major compound from instant coffee extract responsible for graphene green production from graphite and functionalization [33]. 5-CQA is considered as the precursor of caffeic and quinic acids, which may be obtained via hydrolysis reactions during extraction from plant material [34].
Importantly, the derivatives of HCQAs are also widely researched for their potential use in the production of drugs or the use in biopharmaceuticals [35,36]. Moreover, the extract of green coffee beans containing a large amount of 5-CQA has been certified by COSMOS to obtain a label of a natural raw material for cosmetics [37]. It is also produced by Naturex under the trade name Svetol ® as a supplement for body weight loss [38].
This paper aims to discuss and assess the recent advances in chemical synthesis of HCQAs, their extraction from plant material and agricultural waste as well as emerging bioproduction of these compounds using natural or modified micro-organisms. The HCQAs production strategies and relevant research developments are summarized. 5-CQA has been found to be useful for diverse applications. Recently, it has been shown to have potential to counteract SARS-CoV-2 by reducing viral attachment to the host cell-surface heat shock protein A5 (HSPA5) [23]. 5-CQA stimulates short-chain fatty acid production in bacteria. The fermentation products of this acid stimulate the proliferation of Bifidobacterium spp. causing the decreased ratio between Firmicutes and Bacteroidetes [24]. The final compounds formed from 5-CQA and other HCQAs are hippuric acid and 3-hydroxyhippuric acid, which are used as non-specific biomarkers for polyphenol uptake or metabolism [25][26][27].
Moreover, 5-CQA has found applications in the synthesis of metals' nanoparticles as a reducing and stabilizing agent [28,29]; in the production modification of carbon ceramic electrode for NADH detection [30]; in red food dye preparation from coupling of tryptophan and 5-CQA [31]; and the preservation of food by preparing the edible coating with chitosan [32]. This compound is also referred to as the major compound from instant coffee extract responsible for graphene green production from graphite and functionalization [33]. 5-CQA is considered as the precursor of caffeic and quinic acids, which may be obtained via hydrolysis reactions during extraction from plant material [34].
Importantly, the derivatives of HCQAs are also widely researched for their potential use in the production of drugs or the use in biopharmaceuticals [35,36]. Moreover, the extract of green coffee beans containing a large amount of 5-CQA has been certified by COSMOS to obtain a label of a natural raw material for cosmetics [37]. It is also produced by Naturex under the trade name Svetol ® as a supplement for body weight loss [38].
This paper aims to discuss and assess the recent advances in chemical synthesis of HCQAs, their extraction from plant material and agricultural waste as well as emerging bioproduction of these compounds using natural or modified micro-organisms. The HCQAs production strategies and relevant research developments are summarized.

Marine Sources of HCQAs
HCQAs are present in marine sources, mainly microalgae (Spongiochlori sp., Euglena cantabrica, Anabaena doliolum, Porphyra tenera, Undaria pinnatifia) and cyanobacteria (Nostoc sp.) [64][65][66][67]. The HCQAs synthesis pathway in these organisms is similar to higher plants [68]. Although microalgae and cyanobacteria contain high levels of extractable phenolic compounds (phenolic acids) and quinic acid, the abundance of various HCQAs is limited. To date, the data on abundance of 5-CQA is only available for some species of marine plants and cyanobacteria. Its concentration reaches up to 78 µg/g DW for microalgae and 9.55 µg/g DW for cyanobacteria ( Table 3). The higher amount of 5-CQA is determined in algae due to the adaptation to abiotic and biotic stress occurring in the evolutionary advanced micro-organisms [65].

HCQAs Extraction
HCQAs are usually extracted by conventional, solid-liquid extraction (SLE) and nonconventional or intensified techniques, such as ultrasound assisted (UAE), microwave assisted (MAE), pressurized liquid (PLE), supercritical fluid (SFE), enzymatic extraction (EAE) [69,70] or infrared assisted extraction (IAE) [71] (Table 4). Only in EAE, the release and extraction is performed for HCQAs trapped within the plant cell walls using enzymes (cellulases, glucosidases, proteases, dextranases, xylanases and ligninolytic enzymes that do not hydrolase the HCQAs into their constituents) in aqueous buffers or ionic liquids [72][73][74][75]. The non-conventional or intensified extraction methods are considered to be more efficient. In extraction process, parameters (such as temperature, time, pH of solvents, particle size, solvent type and concentration, its volume and other specific parameters of the process (e.g., microwave power, ultrasonic frequency, enzyme concentration)) can be optimized [69,76]. The final yield of HCQAs depends on the raw material as well as the extraction method ( Table 4). The highest yields (up to 4% of raw material) of HCQAs are determined for the coffee by-products, honeysuckle (Lonicera japonicae) and its flowers or by-products, sunflower seed kernels [60,[77][78][79][80][81][82]. All the extraction methods are potentially suitable for the HCQAs except for SFE, which is considered as the green extraction technique, but the recovery yields do not exceed~52%, due to reduced solubility in the nonpolar supercritical fluid [80,83]. The MAE and IRE are usually very efficient and require a short time for the extraction process, but the large size microwave extractors in industry remain too expensive. Therefore, SLE is considered the most relevant method for industrial application due to its simplicity, reproducibility, low cost and possibility to use environment friendly solvents [84]. Intensified methods are mostly applied for the recovery of HCQAs from waste [71,[85][86][87][88][89][90][91][92][93] (Table 4).  Many intensified extraction methods have been adopted for waste valorization. Furthermore, combined processes, such as microwave-assisted simultaneous distillation and dual extraction (MSDDE) [90], multi-frequency multimode modulated vibration (acoustic probe) technique (MMM) [91] or simultaneous SLE extraction and solid-state fermentation (SLE-SSF) [92] have been developed for 5-CQA extraction (Table 4).These three methods are more effective than SLE or other non-conventional extraction methods, however, MSDDE and MMM are too expensive for industrial application, and only SLE-SSF can be applied for large scale extraction and production of 5-CQA with high enrichment of raw material (for example, up to 400% for coffee pulp) [92].
The extraction and identification of HCQAs suffer from some limitations. Firstly, the stability of HCQAs is reduced by the isomerization or destruction reactions under extraction conditions, especially if these conditions are harsh [2,93]. Secondly, transesterification reactions can occur due to the activity of chlorogenate-dependent caffeoyltransferase, when the extraction of fresh plant material is performed in alcohol or alcohol-water mixtures [104]. The transformed products may be mistakenly identified as the new bioactive ingredients of the plant sources [105]. Another problem is the identification of the compounds in the extracts due to the limited number of commercially available standards and similar properties or spectra of HCQAs as summarized in [2,106].
Extraction and purification processes can be more environment-friendly if ionic liquids or eco-friendly natural deep eutectic solvents are used instead of organic solvents. They have a higher affinity to HCQAs than water [107], and it is easy to purify these solvents by distillation and to re-use them for extraction processes [108,109]. Furthermore, the use of ionic liquids can result in a shorter extraction time [75,110]. Generally, the recycling and re-use of the extraction solvents, application of high solid-to-liquid ratio, and reduced processing time helps to lower the total cost of the extraction [100,111].

Chemical Synthesis of HCQAs
The major HCQAs containing hydroxycinnamoyl moieties can be chemically synthesized performing either esterification or condensation reaction between quinic and hydroxycinnamic acids using pyridine or DMAP as homogenous catalyst in organic solvents (dichlormethane or DMF). The reactions with the best total yields are presented in Figure  5.
The highest total yields have been obtained with Sefkow synthesis method for 1-CQA, 3-CQA, 4-CQA or 5-CQA isomers ( Table 5) using esterification reaction between caffeoylchloride derivative and quinic acid derivatives with unprotected OH groups at 1, 3, 4 and/or 5 positions followed by 1-2 steps of hydrolysis reaction of protecting groups [125,126]. The non-protected quinic acid can also be used in the esterification with suitably protected hydroxycinnamoyl derivative, which results in the formation of all mono-substituted HCQAs with moieties at 1-, 3-, 4-and 5-positions [127]. The synthesis of 5-FQA can be obtained from quinic acid ester with malonate and vanillin without any protected hydroxyl group via Knoevenagel condensation reaction when the (E)-double bond is formed (the total yield of 19%) [128]. Independently of the synthesis method, the final crystalline HCQAs can be purified by recrystallization or by a complex procedure (extraction, concentration and chromatographic purification followed by recrystallization) for the improved purity of the final compound [125,126,128]. All the above methods are commonly used in manufacturing, and they are considered cost-effective as the solvents can be recovered by distillation and reused.

Chemical Synthesis of HCQAs
The major HCQAs containing hydroxycinnamoyl moieties can be chemically synthesized performing either esterification or condensation reaction between quinic and hydroxycinnamic acids using pyridine or DMAP as homogenous catalyst in organic solvents (dichlormethane or DMF). The reactions with the best total yields are presented in Figure 5.
The highest total yields have been obtained with Sefkow synthesis method for 1-CQA, 3-CQA, 4-CQA or 5-CQA isomers ( Table 5) using esterification reaction between caffeoylchloride derivative and quinic acid derivatives with unprotected OH groups at 1, 3, 4 and/or 5 positions followed by 1-2 steps of hydrolysis reaction of protecting groups [126,127]. The non-protected quinic acid can also be used in the esterification with suitably protected hydroxycinnamoyl derivative, which results in the formation of all mono-substituted HCQAs with moieties at 1-, 3-, 4-and 5-positions [128]. The synthesis of 5-FQA can be obtained from quinic acid ester with malonate and vanillin without any protected hydroxyl group via Knoevenagel condensation reaction when the (E)-double bond is formed (the total yield of 19%) [129]. Independently of the synthesis method, the final crystalline HCQAs can be purified by recrystallization or by a complex procedure (extraction, concentration and chromatographic purification followed by recrystallization) for the improved purity of the final compound [126,127,129]. All the above methods are commonly used in manufacturing, and they are considered cost-effective as the solvents can be recovered by distillation and reused.  [126] (1); [129] (2); [125] (3-5); and [128] (6). All reactions performed at room temperature.   [127] (1); [130] (2); [126] (3-5); and [129] (6). All reactions performed at room temperature. There are positive and negative aspects of condensation and esterification methods. Higher total yields can be achieved with the esterification method compared to the condensation method. Irrespective of method, the synthesis of di-or tri-HCQAs is more complex than that of mono-HCQAs. Esterification reaction requires the protection/deprotection of active hydroxyl groups because the HCQAs molecules are sensitive to basic and strong acidic or hydrogenative conditions due to the double bond reduction, transesterification or isomerization reactions as well as possible cleavage reactions [129,131,132]. Additionally, esterification may require low or ultralow temperature, solvent system CH 2 Cl 2 /pyridine/DMAP ratio optimization, and long acidic hydrolysis time for the deprotection reaction [126,127,131,132,136], which could cause the decreased yields of HCQAs due to the side reactions. In contrast, the condensation reaction by Knoevenagel involves benzyl aldehydes use of which does not require any protection of reactive hydroxyl groups [137]. 1-HCQAs and di-or tri-substituted HCQAs are more difficult to obtain by both condensation and esterification methods due to increased steric hindrance.
The prices of the major chemical substances for both synthesis methods are presented in Table 6. The Knoevenagel condensation reaction requires benzyl aldehydes, which are currently commercially produced from naphtha. Hydroxycinnamic acids for the esterification reaction are expensive as they are produced mainly by chemical synthesis. Even biotechnological production of vanillin, syringaldehyde or extraction of hydroxycinnamic acids from plant biomass could not reduce the high synthesis costs of HCQAs.

Modified Micro-Organisms
The HCQAs biosynthesis pathways have been developed in E. coli, Saccharomyces cerevisiae, and Pichia pastoris. For the production of 5-CQA from caffeic acid, the E. coli has been engineered by introducing hydroxycinnamoyl-CoA quinate transferase gene HQT from Nicotiana tabacum and 4-coumarate CoA:ligase gene 4CL, which mediates the formation of coenzyme A thioester with hydroxycinnamic acids and deletion of aroD gene, which is required for the conversion of 3-dehydroquinate into 3-dehydroshikimate [145]. This recombinant strain B-101 was able to accumulate 3-dehydroquinate and caffeoyl-3-dehydroquinate, but it was not able to produce 5-CQA in higher concentrations than 16 mg/L. When gene ydiB encoding shikimate/quinate dehydrogenase was overexpressed followed by the vector and cell concentration optimization, then the production up to 450 mg/L in 24 h was achieved from quinate and additionally supplied caffeic acid [145].
The polyculture technique has been applied for 5-CQA production using engineered E. coli. In the two culture technique, E. coli B-102 was inoculated in a filtrated culture mixture of modified E. coli strain B-TP-CA2, which produced caffeic acid from glucose [147]. After 45 h the highest concentration (78 mg/L) of 5-CQA was obtained. Similarly, de novo 5-CQA production using the polyculture technique with three recombinant E. coli strains containing biosynthetic modules of caffeic acid, quinic acid and 5-CQA, reached 250 µM (or~88 mg/L) concentration after 18 h of incubation [148]. In both cases, a rather low yield of targeted compound could be acceptable due to the usage of a low-cost primary compound (glucose).
Yeast has also been engineered for the synthesis of HCQAs. The expression of tobacco 4CL and globe artichoke HCT genes in yeast Saccharomyces cerevisiae resulted in the formation of N-(E)-p-coumaroyl-3-hydroxyanthranilic acid as a primary product, which is similar to avenanthramides [149]. Subsequently, a successful biosynthesis of 5-CQA and 5-pCoQA in yeast was achieved by expressing a BAHD enzyme NtHQT from tobacco Nicotiana tabacum and 4CL5 [150]. Recently, it has been shown that GDSL lipase-like ICS enzyme from Ipomoea batatas can be used for the efficient conversion of 5-CQA into 3,5-diCQA in P. pastoris [46]. 4CL, HST, and HQT resulted in the biosynthesis of 109.7 mg/L 5-CQA from glucose [145] ( Figure 6). The polyculture technique has been applied for 5-CQA production using engineered E. coli. In the two culture technique, E. coli B-102 was inoculated in a filtrated culture mixture of modified E. coli strain B-TP-CA2, which produced caffeic acid from glucose [146]. After 45 h the highest concentration (78 mg/L) of 5-CQA was obtained. Similarly, de novo 5-CQA production using the polyculture technique with three recombinant E. coli strains containing biosynthetic modules of caffeic acid, quinic acid and 5-CQA, reached 250 µM (or ~88 mg/L) concentration after 18 h of incubation [147]. In both cases, a rather low yield of targeted compound could be acceptable due to the usage of a low-cost primary compound (glucose).
Yeast has also been engineered for the synthesis of HCQAs. The expression of tobacco 4CL and globe artichoke HCT genes in yeast Saccharomyces cerevisiae resulted in the formation of N-(E)-p-coumaroyl-3-hydroxyanthranilic acid as a primary product, which is similar to avenanthramides [148]. Subsequently, a successful biosynthesis of 5-CQA and 5-pCoQA in yeast was achieved by expressing a BAHD enzyme NtHQT from tobacco Nicotiana tabacum and 4CL5 [149]. Recently, it has been shown that GDSL lipase-like ICS enzyme from Ipomoea batatas can be used for the efficient conversion of 5-CQA into 3,5-diCQA in P. pastoris [46]. copy number of 5-CQA pathway genes encoding hydroxycinnamoyl-CoA quinate transferase 2 (HQT2) and cytochrome P450 98A3 (C3′H) [150]. The engineered S. cerevisiae strain produced 5-CQA to 234.8 ± 11.1 mg/L in shake flask culture and 806.8 ± 1.7 mg/L in fed-batch fermentation [150].  Figure 6. The synthesis of chlorogenic acid in E. coli (a) and S. cerevisiae (b) from carbon source according to [146; 151]. Black and blue arrows correspond to native and non-native pathways, respectively. Dashed arrows represent the complex processes. The blue and red names of genes correspond to inserted and overexpressed genes, respectively. Abbreviations: AroH -phospho-

Conclusions
HCQAs are becoming very important compounds in cosmetics, medicine and food supplements production due to their outstanding properties. Therefore, the need of HCQAs, especially 5-CQA, is constantly increasing. The main strategies to obtain these compounds are based on their extraction from plant biomass and chemical synthesis. Recently, biosynthesis using modified or non-modified micro-organisms has attracted significant research efforts.

Conclusions
HCQAs are becoming very important compounds in cosmetics, medicine and food supplements production due to their outstanding properties. Therefore, the need of HC-QAs, especially 5-CQA, is constantly increasing. The main strategies to obtain these compounds are based on their extraction from plant biomass and chemical synthesis. Recently, biosynthesis using modified or non-modified micro-organisms has attracted significant research efforts.
Several conventional and intensified methods have been developed for extraction of HCQAs from plants or marine biomass. Amongst the most promising are solid-liquid extraction techniques (SLE), pressurized liquid (PLE), supercritical fluid (SFE), enzymatic extraction (EAE), even if SLE remains dominant. The successful application of other intensified methods, such as microwave-assisted simultaneous distillation and dual extraction (MSDDE), multi-frequency multimode modulated vibration (acoustic probe) technique (MMM) or simultaneous SLE extraction and solid-state fermentation (SLE-SSF), was demonstrated with some evident success for the 5-CQA. Their suitability for other HCQAs remains to be explored. Despite that, intensified methods can result in better yields and can require less time; however, they are expensive, which limits their industrial application. 5-CQA extraction has already reached the stage of industrialization. 5-CQA is mainly produced (>75%) from honeysuckle, eucommia and green coffee bean. For the less abundant HCQAs, such as SQAs (which is present at extremely low yields in plants), further improvements of the extraction methodology are required.
Chemical synthesis enables achievement of moderate yields of mono-, di-or tri-HCQAs. It requires large quantities of hydroxycinnamic acid and is based on use of condensation or esterification methods. Esterification enables achievement of a better yield, but the high costs of reagents limits use of this method. Overall, the chemical synthesis of HCQAs is expensive and environmentally unfriendly because the primary compounds of the synthesis are obtained mainly from naphtha and its refinery products. Moreover, the use of halogenized organic compounds generates toxic waste. Despite this, this method could be preferable for the production of naturally scarce SQAs or to synthesize HCQAs that are not obtainable by other methods.
Biotechnological production of HCQAs is considered a most promising approach that enables consolidation of green chemistry and circular economy objectives. Despite its early stage, several non-modified micro-organisms have already been shown to produce the CQAs to yields that are comparable to those obtained by extraction from plant biomass. Significantly, the engineered bacteria Escherichia coli or yeast Saccharomyces cerevisiae have been developed to synthesize a greater variety of HCQAs, such as 5-CQA, 5-FQA, 5-p-CoQA from simple carbon sources. The 5-CQA titer of approximately 0.8 g/l has been achieved in fed-batch fermentation using Saccharomyces cerevisiae. Although the biotechnological production of HCQAs requires a significant advancement to make it suitable for industrial application, the utilization of micro-organisms shows great promise in the recycling and recovery of HCQAs from organic waste.